1. Field of the Invention
The present invention relates generally to the field of viral vectors and the use of such vectors to express foreign DNA in mammalian cells. The invention also relates to the field of gene therapy and particularly gene therapy involving viral vectors to direct genetic material to be expressed in particular tissues. More particularly, it concerns adenovirus and the ability to displace a large amount of the adenoviral genome with heterologous DNA and to replicate the viral construct in a helper cell line and express the foreign DNA in a host cell.
2. Description of the Related Art
Gene therapy is an area that offers an attractive alternative for the treatment of many diseases and disorders. In particular, the ability of viruses to enter a cell and express its genetic material in the host cell raises the possibility of replacing lost or defective gene function in vivo. However, for gene therapy to succeed, there is a need for new vectors with the properties of high therapeutic index, large capacity, targeted gene delivery, and tissue-specific gene expression. High therapeutic index indicates high therapeutic effect with low adverse effect, or in other words, a high rate of cure or improvement of the disorder with low or no side effects. This is a pharmacological concept that applies to all therapeutic agents, including gene therapy vectors. However, currently available gene transfer vectors are not able to meet the requirement of high therapeutic index (Mulligan, 1993), because of the vector-borne cycto- or geno-toxicities that are associated with many of the current gene transfer vectors.
Multiple and targeted gene transfer is particularly relevant to gene therapy for cancer (Friedmann, 1992). Throughout the last decade, studies of oncogenes and tumor suppressor genes have revealed more and more evidence that cancer is a disease developed through a process of multiple cytogenetic disorders (Chiao et al., 1990; Levine, 1990; Weinberg, 1991; Sugimura et al., 1992). Based on this concept of carcinogenesis, new strategies have developed rapidly as alternatives to conventional cancer therapy (Renan, 1990; Lotze et al., 1992; Pardoll, 1992). One of these is gene therapy (Friedmann, 1989), in which tumor suppressor genes, antisense oncogenes, and other related genes are used as therapeutic genes. Some strategies contemplated for gene therapy of cancer are the restoration of tumor-suppressor gene function, the blocking of oncogene expression and the correction of other gene-related disorders in cancer cells. It is believed that to achieve a maximal therapeutic effect, targeted delivery of a combination of these therapeutic genes by a single higher-capacity vector into cancer cells will be essential. Unfortunately, no such vector is currently available.
Another example of a disease for which a large capacity vector might be effective is Duchenne muscular dystrophy (DMD), a lethal, X-linked degenerative disorder of muscle, which affects about 1 in 35,000 newborn males. DMD is caused by a deficiency of dystrophin (Zubrzycka-Gaarn et al., 1988), a 427 kD protein encoded by a 14-kb transcript (Koenig et al., 1987). A possible therapy for this disease would be the restoration of dystrophin function by insertion and expression of the dystrophin gene in the patient's muscles (Blau, 1993; Cox et al., 1993). This therapy would require a vector that could efficiently deliver the 14 kb cDNA into muscle cells and specifically express the DMD protein in the muscle cells. Unfortunately, at the time of this disclosure, there is no vector system available which is capable of delivering more than 7.5 kb of DNA to be expressed in a specific tissue.
Retroviruses were the earliest gene transfer vectors. They were first used to insert gene markers and for transducing the cDNA of adenosine deaminase (ADA) into human lymphocytes (Miller, 1992). Unfortunately, retroviruses have several drawbacks as gene therapy agents, including genotoxicity caused by integration into the host genome, instability, dependence on target cell receptors for infection and proliferation only in actively dividing cells (Miller and Rosman, 1989; Major, 1992). In addition, the maximum gene-carrying capacity of retroviral vectors is under 10 kb (Morgenstern and Land, 1991).
Adeno-associated virus (AAV) has recently been developed as a gene transfer system. Wild-type AAV has high infectivity and specificity in integrating into the host cell genome (Hermonat and Muzyczka, 1984; Lebkowski et al., 1988). However, experimental data has shown that recombinant AAV tend to have low titers and lose their specificity of integration (Samulski et al., 1989). Also, the maximum gene-carrying capacity for AAV is under 5 kb (Walsh et al., 1992).
Herpes simplex virus type-1 (HSV-1) is attractive as a vector for applications directed to the nervous system because of its neurotropic property (Geller and Federoff, 1991; Geller, 1993). The HSV-1 genome has over 70 genes located along a 150-kb DNA molecule (Roizman and Sears, 1990), but the largest foreign DNA insertion in the virus has been 7 kb (Knipe et al., 1978). For example, a helper cell line (E5 cells) available for the propagation of replication-defective HSV-1 has complemented the 5 kb ICP4 gene (Deluca et al., 1985). However, because of its very complex genome, a much greater understanding of the interaction of HSV with host cells is required before a suitable HSV vector system can be engineered. In particular, an appropriate vector backbone has not been developed and issues related to gene expression during latency have not been resolved (Glorioso et al., 1992).
Vaccinia virus, from the poxvirus family, has also been developed as an expression vector (Moss, 1991; Moss, 1992). The vaccinia genome is among the most complex of all animal viruses, comprising approximately 200 discrete protein-coding regions along a nearly 200-kb DNA molecule (Goebel, et al., 1990). It has been shown that approximately 25 kb of foreign DNA could be inserted into the viral genome and packaged into the virion (Smith and Boss, 1983). However, the extreme cytotoxicity of vaccinia virus presents a limitation to its use in gene therapy applications. Until this is overcome, vaccinia virus will not be suitable for in vivo gene therapy. Other potential viral vectors exist (Mulligan, 1993; Kriegler, 1990), but they either are not well characterized or do not have the necessary characteristics for a supervector system.
Adenoviruses have been widely studied and well-characterized as a model system for eukaryotic gene expression. Ad are easy to grow and manipulate, and they exhibit broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1011 plaque-forming unit (PFU)/ml, and they are highly infective. The life cycle of Ad does not require integration into the host cell genome. The foreign genes delivered by Ad vectors are expressed episomally, and therefore, have low genotoxicity to host cells. Ad appear to be linked only to relatively mild diseases, since there is no known association of human malignancies with Ad infection. Moreover, no side effects have been reported in studies of vaccination with wild-type Ad (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Ad vectors have been successfully used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies demonstrated that recombinant Ad could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Successful experiments in administering recombinant Ad to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injection (Herz and Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).
Generation and propagation of the current Ad vectors depend on a unique helper cell line, 293, which was transformed from human embryonic kidney cells by AD5 DNA fragments and constitutively expresses E1 proteins (Graham, et al., 1977). Since the E3 region is dispensable from the Ad genome (Jones and Shenk, 1978), the current Ad vectors, with the help of 293 cells, carry foreign DNA in either the E1, the E3 or both regions (Graham and Prevec, 1991). In nature, Ad can package approximately 105% of the wild-type genome (Ghosh-Choudhury, et al., 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current Ad vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the Ad viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1 deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available Ad vectors at high multiplicities of infection (Mulligan, 1993).
Another problem with the currently available vectors is the potential for generation of wild-type virus by recombination. This may occur because the left end of the current Ad vectors has a sequence of about 1.5 kb (9.8-14 mu) overlapping with the E1 fragment in 293 cells (Graham, et al., 1977). Homologous recombinations that generate wild-type virus were detectable when E1 substitution vectors were extensively amplified in 293 cells (personal communication, Dr. Richard Gregory, CANJI, Inc., San Diego, Calif.).
Finally, Ad mutants with deletions at different regions of the viral genome have been rescued by helper viruses that provide the deleted gene products in trans. In cell line W162 (Weinberg and Ketner, 1983), which was stably transfected with the E4 region DNA, the constantly expressed E4 proteins supported propagation of E4 deletion (92-99 mu) mutants (Bridge et al., 1993). However, it has not been believed possible to delete the large E2 region which would provide for the insertion of up to 35 kb of foreign DNA into the adenoviral vector.
Therefore, there still exists an immediate need for an adenoviral supervector system which will have a high therapeutic index, a large carrying capacity of heterologous DNA and the capacity for targeted gene delivery and tissue specific expression. Such a vector system will have utility in a wide variety of in vivo and in vitro applications such a gene therapy protocols, the production of useful protein products in mammalian cell culture, as gene transfer markers or for the diagnosis of genetic deficiencies in particular cell lines.